Construction of silk fiber core in lepidoptera

The formation and properties of lepidopteran silk fibers depend on amino acid repeats in the principal protein, heavy chain fibroin (H-fibroin). In H-fibroins of the "bombycoid" type, concatenations of alanine or of the GAGAGS crystalline motifs (1st tier repeats) and adjacent sequences breaking periodicity make 2nd tier repeats. Two to six such repeats comprise a 3rd tier assembly, and 12 assemblies, linked by an amorphous sequence, constitute the repetitive H-fibroin region. Heterogeneity in the repeat length and intercalation of amorphous regions prevent excessive crystallization. In the "pyraloid" H-fibroins, iterations of simple motifs are absent and assemblies of several complex motifs constitute highly regular repeats that are organized in about 12 highest order reiterations without specific spacers. Repeat homogeneity appears crucial for the alignment and interaction of the disjunct motifs that must be registered precisely to form crystallites; repeat heterogeneity is associated with decreased fiber strength. Both H-fibroin types are typically hydrophobic, and their secretion requires disulfide linkage to light chain fibroin and participation of another protein, P25. These auxiliary proteins are absent in saturniid moths with amphiphilic H-fibroin repeats. The selection at nucleic acid and protein levels and the availability of nutrients play roles in H-fibroin evolution.     

The Design of Silk Fiber Composition in Moths Has Been Conserved for More Than 150 Million Years

The silk of caterpillars is secreted in the labial glands, stored as a gel in their lumen, and converted into a solid filament during spinning. Heavy chain fibroin (H-fibroin), light chain fibroin (L-fibroin), and P25 protein constitute the filament core in a few species that have been analyzed. Identification of these proteins in Yponomeuta evonymella, a moth from a family which diverged from the rest of Lepidoptera about 150 million years ago, reveals that the mode of filament construction is highly conserved. It is proposed that association of the three proteins is suited for long storage of hydrated silk dope and its rapid conversion to filament. Interactions underlying these processes depend on conserved spacing of critical amino acid residues that are dispersed through the L-fibroin and P25 and assembled in the short ends of the H-fibroin molecule. Strength, elasticity, and other physical properties of the filament are determined by simple amino acid motifs arranged in repetitive modules that build up most of the H-fibroin. H-Fibroin synergy with L-fibroin and P25 does not interfere with motif diversification by which the filament acquires new properties. Several types of motifs in complex repeats occur in the silks used for larval cobwebs and pupal cocoons. Restriction of silk use to cocoon construction in some lepidopteran families has been accompanied by simplification of H-fibroin repeats. An extreme deviation of the silk structure occurs in the Saturniidae silkmoths, which possess modified H-fibroin and lack L-fibroin and P25. [Reviewing Editor: Dr. David Pollok]     

Protein composition of silk filaments spun under water by caddisfly larvae

Silk fiber produced by the larvae of Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) is composed of two filaments embedded in a layer of glue proteins. In an aerial environment Lepidoptera spin silk filaments assembled from heavy chain fibroin (H-fibroin), light chain fibroin (L-fibroin), and the glycoprotein P25. The silk filament of caddisflies, which is produced and persists in water, contained homologues of H-fibroin (>500 kDa) and L-fibroin (25 kDa) but not of P25. The amphiphilic nature of H-fibroin and its high content of charged amino acids probably facilitate the secretion and storage of a covalently linked L-fibroin/H-fibroin dimer in the absence of P25. Several types of short amino acid motifs were arranged in orderly fashion in the regularly reiterated repeats that made up more than 95% of the length of H-fibroin. The H-fibroins of Hydropsyche angustipennis and Limnephilus decipiens from different caddisfly suborders contained GPXGX, SXSXSXSX, and GGX motifs such as the lepidopteran and spider silks but differed from them by a lack of poly(A) and poly(GA) motifs. H-fibroins of both caddisfly species harbored a conserved repeat of 31 residues but were distinguished by a few species-specific motifs and their organization in higher order repeats. Structural differences may be related to the silk function as a catching net in H. angustipennis and a stitching fiber in L. decipiens.     

Unique molecular architecture of silk fibroin in the waxmoth, Galleria mellonella

Proteins of silk fibers are characterized by reiterations of amino acid repeats. Physical properties of the fiber are determined by the amino acid composition, the complexity of repetitive units, and arrangement of these units into higher order arrays. Except for very short motifs of 6-10 residues, the length of repetitive units and the number of these units concatenated in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes an exceptional silk protein represented by the 500-kDa heavy chain fibroin (H-fibroin) of the waxmoth, Galleria mellonella. Its non-repetitive N-terminal (175 residues) and C-terminal (60 residues) parts, the overall gene organization, and the nucleotide sequence around the TATA box show that it is homologous to the H-fibroins of other Lepidoptera. However, over 95% of the protein consists of highly ordered repetitive structures that are unmatched in other species. The repetitive region includes 11 assemblies AB(1)AB(1)AB(1)AB(2)(AB(2))AB(2) of remarkably conserved polypeptide repeats A (63 amino acid residues), B(1) (43 residues), and B(2) (18 residues). The repeats contain a high proportion of Gly (31.6%), Ala (23.8%), Ser (18.1%), and of residues with long hydrophobic side chains (16% for Leu, Ile, and Val combined). The presence of the GLGGLG and SSAASAA(AA) motifs suggests formation of pleated beta-sheets and their stacking into crystallites. Conspicuous conservation of the apolar sequence VIVI followed by DD or ED is interpreted as indicating the importance of hydrophobicity and electrostatic charge in H-fibroin cross-linking. The environment of G. mellonella larvae within bee cultures requires continuous production of silk that must be both strong and elastic. The spectacular arrangement of the repetitive H-fibroin region apparently evolved to meet these requirements.     

Evidence for an immune function of lepidopteran silk proteins

Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.     

Venturia canescens parasitizing Galleria mellonella and Anagasta kuehniella: differing suitability of two hosts with highly variable growth potential

Venturia canescens (Grav.) (Hymenoptera: Ichneumonidae) is a solitary larval koinobiont endoparasitoid, ovipositing in several larval instars of different pyralid moth species that are pests of stored food products. After oviposition, the host larva continues to feed and grow for at least several days, the precise time doing so depending on the stage attacked. We examined the relationship between host stage and body mass on parasitoid development in late second to fifth instars of two hosts with highly variable growth potential: the wax moth, Galleria mellonella (L) and the flour moth, Anagasta kuehniella (Zeller)(Lepidoptera: Pyralidae). G. mellonella is the largest known host of V. canescens, with healthy larvae occasionally exceeding 400mg at pupation, whereas those of A. kuehniella rarely exceed 40 mg at the same stage. Parasitoid survival was generally higher in early instars of G. mellonella than in later instars. By contrast, percentage adult emergence in A. kuehniella was highest in late fifth instar and lowest in late second instar. A. kuehniella was the more suitable host species, with over 45% adult emergence in all instars, whereas in G. mellonella we found less than 35% adult emergence in all instars. Adult parasitoid size increased and egg-to-adult development time decreased in a host size- and instar-specific manner from A. kuehniella. The relationship between host size and stage and these fitness correlates was less clear in G. mellonella. Although both host species were parasitized over a similar range of fresh weights, the suitability weight-range of A. kuehniella was considerably wider than G. mellonella for the successful development of V. canescens. However, in hosts of similar weight under 5 mg when parasitized, larger wasps emerged from G. mellonella than from A. kuehniella. Parasitoid growth and development is clearly affected by host species, and we argue that patterns of host utilization and resource acquisition by parasitoids have evolved in accordance with host growth potential and the nutritional requirements of the parasitoid.     

Moth sex chromatin probed by comparative genomic hybridization (CGH).

Abstract: Comparative genomic hybridization (CGH) with a probe mixture of differently labeled genomic DNA from females and males highlighted the W chromosomes in mitotic plates and the W chromatin in polyploid interphase nuclei of the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella. The overproportionate fluorescence signal indicated an accumulation of repetitive sequences in the respective W chromosomes. Measurements of the fluorescence signals revealed two components, one that is present also in male DNA (non-W chromosomes) and another one that is present only in or preponderantly in female DNA (W chromosomes). While the W chromosomes of E. kuehniella and G. mellonella had both components, that of B. mori appeared to lack the latter component. Our results show that CGH can be applied to obtain a first estimate of the sequence composition of sex chromosomes in species from which otherwise little is known on the molecular level.     

Characterization of unique heavy chain fibroin filaments spun underwater by the caddisfly <Emphasis Type="Italic">Stenopsyche marmorata</Emphasis> (Trichoptera; <Emphasis Type="Italic">Stenopsychidae</Emphasis>)

The silks of both Lepidoptera and its sister order Trichoptera contain a homologue of heavy chain (H-fibroin), which is assumed to determine the physical properties of the fiber, such as elasticity and toughness. The long repetitive region of the H-fibroin caddisfly Stenopsyche marmorata shows a conspicuous hierarchical structure that is composed of huge units, which are mainly constructed from four large blocks (SA, SB, SC and SD) arranged in an orderly fashion. Each block contains short, distinct motifs such as SXSXSX(SX), GPXG(X)_1–3 or triplet GGX, which also occur in lepidopteran and spider filaments. The SA, SB and SC blocks have nearly fixed amino acid numbers, while the length of the SD block varies, usually due to a variable number of GPXGXXX repeats. The multiple sandwich structure that occurs in the SB block is assumed to be unique to the caddisfly and may be related to the use of silk in an aqueous environment. The overall average of hydrophilicity in the repetitive H-fibroin region of S. marmorata is −0.609, whereas hydrophobicity prevails in most lepidopteran H-fibroins. Gly (29.51%), Pro (11.28%) and Ser (10.90%) are the three predominant amino acids of H-fibroin, and the high content of essential amino acids reflects the energy-rich food resources of the caddisfly. The H-fibroin of S. marmorata is about 400–500 kDa and expressed in both the middle and posterior silk glands, which is different from the expression pattern in Lepidoptera species.     

Is the sex communication of two pyralid moths, Plodia interpunctella and Ephestia kuehniella, under circadian clock regulation?

Females of the Indian meal moth, Plodia interpunctella, and females of the Mediterranean flour month, Ephestia kuehniella (both Lepidoptera: Pyralidae), exhibit daily rhythms in calling behavior. The peak in P. interpunctella calling occurs at dusk, whereas E. kuehniella calls preferentially at dawn. This behavior turned arrhythmic in P. interpunctella females in constant darkness (DD) and remained arrhythmic in constant light (LL), whereas E. kuehniella females showed a persistent rhythm in DD and suppression of the behavior in LL, indicating regulation by a circadian clock mechanism. The rhythm of male locomotor activity corresponded well with the sexual activity of females, reaching the peak at dusk in P. interpunctella and at dawn in E. kuehniella. An immunohistochemical study of the pheromone biosynthesis activating neuropeptide, corazonin, and pigment dispersing factor revealed distinct sets of neurons in the brain-subesophageal complex and in the neurohemal organs of the 2 species.     

Mechanism of secretion of the contents of the mandibular glands of Plodia interpunctella larvae

ABSTRACT. Larvae of Plodia interpunctella deposit droplets of mandibular gland secretion onto silk filaments connecting particles within the substrate they inhabit, and on to the substrate itself. Active participation of the mouthparts is necessary for both the formation and deposition of these droplets along the filaments and occurs as the silk is spun. Deposition of secretion directly on the substratum does not involve the mouthparts and appears to result from contact between the lower surface of the head and the substratum. Larvae from which the mandibular glands have been removed by excision neither produce droplets on silk nor deposit them on the substratum. Those which are incapable of spinning silk due to cauterization of the spinneret are still able to deposit secretion on the substratum, however. Deposition of secretion on silk webbing does not occur when the setae which regulate formation of droplets on filaments are removed by shaving. Droplets are not characteristic of a particular larval instar but are produced by larvae of all instars as a normal function which does not depend upon intraspecific encounters. Four other Lepidopteran pests, Ephestia elutella, Ephestia cautella, Anagasta kuehniella and Antigasta catolaunalis , were also found to produce droplets of mandibular gland secretion in a similar manner to larvae of P. interpunctella.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Silk tape nanostructure and silk gland anatomy of trichoptera

Caddisflys (order Trichoptera) construct elaborate protective shelters and food harvesting nets with underwater adhesive silk. The silk fiber resembles a nanostructured tape composed of thousands of nanofibrils (～ 120 nm) oriented with the major axis of the fiber, which in turn are composed of spherical subunits. Weaker lateral interactions between nanofibrils allow the fiber to conform to surface topography and increase contact area. Highly phosphorylated (pSX)(4) motifs in H-fibroin blocks of positively charged basic residues are conserved across all three suborders of Trichoptera. Electrostatic interactions between the oppositely charged motifs could drive liquid-liquid phase separation of silk fiber precursors into a complex coacervates mesophase. Accessibility of phosphoserine to an anti-phosphoserine antibody is lower in the lumen of the silk gland storage region compared to the nascent fiber formed in the anterior conducting channel. The phosphorylated motifs may serve as a marker for the structural reorganization of the silk precursor mesophase into strongly refringent fibers. The structural change occurring at the transition into the conducting channel makes this region of special interest. Fiber formation from polyampholytic silk proteins in Trichoptera may suggest a new approach to create synthetic silk analogs from water-soluble precursors.     

柞蚕热休克蛋白70基因的克隆和序列分析

HSP70蛋白是受热等因素刺激后而诱导产生的蛋白质,是热休克蛋白家族中最重要的一员。采用RT-PCR方法克隆了柞蚕(Antheraea pernyi)热休克蛋白70基因(HSP70)的ORF序列(GenBank登录号:GU945199),该片段的序列长度为1905bp。生物信息学分析表明,该序列共编码634个氨基酸,预测蛋白的等电点和分子量大小分别为5.62kD和69.5kD。具有HSP70的保守性结构特征,与天蚕(Antheraea yamamai)、家蚕(Bombyx mor)、甘蓝夜蛾(Mamestra brassicae)、棉铃虫(Heliothis viriplaca)、甜菜夜蛾(Spodoptera exigua)、烟草夜蛾(Manduca sexta1)、膜翅目寄生蜂(Cotesia rubecula)的同源性分别为95.7%、78.5%、76.1%、77.3%、76.6%、74.7%、65.9%。根据它们的一级结构构建了系统进化树,进一步确立了它们之间的亲缘关系。     

Differential distribution of simple sequence repeats in eukaryotic genome sequences.

Complete chromosome/genome sequences available from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae were analyzed for the occurrence of mono-, di-, tri-, and tetranucleotide repeats. In all of the genomes studied, dinucleotide repeat stretches tended to be longer than other repeats. Additionally, tetranucleotide repeats in humans and trinucleotide repeats in Drosophila also seemed to be longer. Although the trends for different repeats are similar between different chromosomes within a genome, the density of repeats may vary between different chromosomes of the same species. The abundance or rarity of various di- and trinucleotide repeats in different genomes cannot be explained by nucleotide composition of a sequence or potential of repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication/repair/recombination machinery might play an important role in the genesis of repeats. Moreover, analysis of complete genome coding DNA sequences of Drosophila, C. elegans, and yeast indicated that expansions of codon repeats corresponding to small hydrophilic amino acids are tolerated more, while strong selection pressures probably eliminate codon repeats encoding hydrophobic and basic amino acids. The locations and sequences of all of the repeat loci detected in genome sequences and coding DNA sequences are available at http://www.ncl-india.org/ssr and could be useful for further studies.     

Identification and analysis of novel tandem repeats in the cell surface proteins of archaeal and bacterial genomes using computational tools

We have identified four novel repeats and two domains in cell surface proteins encoded by the Methanosarcina acetivorans genome and in some archaeal and bacterial genomes. The repeats correspond to a certain number of amino acid residues present in tandem in a protein sequence and each repeat is characterized by conserved sequence motifs. These correspond to: (a) a 42 amino acid (aa) residue RIVW repeat; (b) a 45 aa residue LGxL repeat; (c) a 42 aa residue LVIVD repeat; and (d) a 54 aa residue LGFP repeat. The domains correspond to a certain number of aa residues in a protein sequence that do not comprise internal repeats. These correspond to: (a) a 200 aa residue DNRLRE domain; and (b) a 70 aa residue PEGA domain. We discuss the occurrence of these repeats and domains in the different proteins and genomes analysed in this work.     

Homologues of fibroin L-chain and P25 of Bombyx mori are present in Dendrolimus spectabilis and Papilio xuthus but not detectable in Antheraea yamamai

Low molecular mass protein components of fibroin, whose electrophoretic patterns before and after the reductive cleavage of disulfide bonds were similar to those of L-chain and P25 of Bombyx mori, were identified in fibroin samples of Bombyx mandarina, Dendrolimus spectabilis and Papilio xuthus but not of Antheraea yamamai. Fibroin of A. yamamai is suggested to form a dimer of H-chain. Full length cDNA sequences were cloned for the homologues of L-chain and P25 from B. mandarina, D. spectabilis and P. xuthus. The deduced sequences of L-chain and P25 of B. mandarina are almost identical to those of B. mori, each containing a single amino acid change. Homologues of L-chain and P25 of D. spectabilis and P. xuthus show about 50% overall identity, respectively, with those of B. mori, but essential structural features; i.e. the three Cys residues in an L-chain and the eight Cys residues and one of the potential N-glycosylation sites in P25, are conserved in both species. These results, together with the published results for Galleria mellonella, suggest that the three-components (H-chain, L-chain and P25) complex of fibroin is rather common among Lepidopteran silk-producing insects, in contrast to the H-H dimer type found in the saturnid silkworm.